In aplastic anemia, the bone marrow is replaced by fat, and peripheral blood counts - - of white blood cells, red blood cells, and platelets - - fall to extremely low levels, leading to death from anemia, bleeding or infection. Aplastic anemia is a disease of young persons and in its severe form is almost invariably fatal untreated. Historically, aplastic anemia has been linked to chemical exposures, in particular benzene; it is an idiosyncratic complication of some medical drug use; it occurs as a rare event in pregnancy and following seronegative hepatitis; and the diseases associated with certain immunologic conditions. The chance observation that some patients post-bone marrow transplant recovered their own marrow function led to the inference that the immunosuppressive conditioning regimen might have treated an underlying immune-mediated pathophysiology. Purposeful administration of antithymocyte globulin (ATG) has led to hematologic recovery in the majority of treated patients. Laboratory data have also revealed abnormalities of the immune system: lymphocyte populations that induce apoptosis in hematopoietic target cells by the Fas-mediated pathway, and oligoclones of effector T cells which express type 1 cytokines, especially gamma-interferon. The Hematology Branch has been a leader in both the scientific and medical studies of aplastic anemia pathophysiology and treatment. Several major clinical protocols have been completed and published. These include studies in which we determined a) prolonged cyclosporine administration does not prevent a relapse, but a low drug dose may be adequate to maintain blood counts; b) horse ATG after failed rabbit ATG therapy is generally ineffective; and c) moderate dose cyclophosphamide is toxic and, while clinically active in restoring hematopoiesis, is not superior to standard horse ATG plus cyclosporine. These negative studies have prompted novel approaches to improve the response and outcomes with horse ATG and cyclosporine obtained in our institution and many others (about 65% response rate, 35% relapse rate, and 15% evolution to MDS/AML). As described also in Dr. Dunbars annual report, we were surprised to find that approximately 40% of patients with refractory severe aplastic anemia, who had failed multiple previous treatments and were chronically transfused red blood cells and platelets, responded to eltrombopag, a thrombopoeitin mimetic which is administered orally. Responses were often bi-and trilineage, robust with marked improvement blood counts, and accompanied by increased bone marrow cellularity. These startling results, which have led to accelerated approval by the FDA of a new indication of eltrombopag based on our investigator-initiated single institution pilot studies, have led to multiple protocols testing eltrombopag in bone marrow failure. For refractory disease, an extension study, confirmed the initial response rate and suggested that patients who do respond can have eltrombopag discontinued without relapse. However, the rate of clonal evolution, as manifested by cytogenetic abnormalities, may be higher than expected in refractory patients who are treated long-term with eltrombopag. Eltrombopag as a single agent has also produced about a 40% response rate in patients with moderate aplastic anemia and in low risk myelodysplastic syndrome. For the treatment of aplastic anemia patients on presentation, we have added eltrombopag to standard immunosuppressive therapy in an anticipated three stage series of protocol. In the initial group of 31 patients (30 actually treated), eltrombopag raised the overall response rate to about 80% and speeded hematologic improvement. The rate of clonal evolution was low. The initial stage paired eltrombopag with immunosuppression and dictated a six month treatment period for experimental drug. Although there were no unexpected or serious toxicities, it is desirable to reduce drug exposure because of the risk of clonal evolution, in a second stage study, now underway, eltrombopag is administered for only three months. A third phase study is in planning, in which eltrombopag and cyclosporine would be implemented urgently, with ATG added when Fanconi anemia and other cytogenetic testing is completed (these tests often require several weeks). The third stage would promote early stem cell recovery and rapid institution of immunosuppression, identify any risk of shared hepatotoxicity with the two oral drugs, and model eltrombopag and cyclosporine alone as developing world treatment for severe aplastic anemia. The mechanism of action of eltrombopag is likely stem cell stimulation, based on a variety of tissue culture, animal and human data. In the treatment-nave protocol, CD34 cells have been measured pre- and post-treatment, and they increased dramatically, almost 30-fold. Furthermore, hematopoietic progenitors become detectable in most inpatients, as determined by sophisticated flow cytometric phenotyping. There may be additional effects on immune components, especially T-regulatory cell activation. In our basic research laboratory, we have explored the mechanisms of immune-mediated marrow failure in our well established rodent model. Signal advances in this area include studies of T-regulatory cells as protective of the hematopoietic stem niche, adipocytes as repressors as inhibitors of hematopoiesis, and the precise role of gamma interferon in both immune and hematopoietic cell regulation. We were unable to confirm a prominently published paper claiming that hematopoietic cells in mouse models can persist in immunologically protected niches, due to the presence of T-regulatory cells. We were unable to confirm that adipocytes act as negative regulators of hematopoiesis: in extensive experiments, they appear to function in this role only in the setting of immune mediated bone marrow failure, and the purportedly specific inhibitors of adipogenesis also act to repress the cytotoxic immune response. We have revisited the role of gamma interferon in hematopoiesis. We confirmed the results of others that gamma interferon in the mouse can increase the quantity of hematopoietic cells and lead to their exit from the stem cell niche; however, these cells are not functionally active in competitive repopulation assays. Furthermore, gamma interferon, as we and others have previously described, increases Fas expression on hematopoietic stem cells and in the presence of cytotoxic CD8 lymphocytes leads to apoptosis in this population. Therefore, gamma interferons effects on hematopoiesis are context-dependent. We have extended our studies of immune mediated bone marrow failure in a murine model by establishing conditions for donor lymphocytes and irradiation in B6 mice, which has allowed us to examine transgenic and knock-out effects as this is the genetic background to produce such animals. As an example, for gamma interferon knock-out donor lymphocytes are relatively inactive in promoting aplastic anemia in this model, consistent with the hypothesized role gamma interferon as a critical regulator of immune destruction of hematopoietic cells in the mouse and in humans.